1. Field of the Invention
The invention relates to a novel lactam ring-opening enzyme, and to a use thereof. More specifically, the invention relates to a novel lactam ring-opening enzyme having the activity of opening the lactam ring of a streptothricin derivative, to a method for manufacturing a lactam ring-opened streptothricin derivative using this enzyme, and to an antimicrobial agent containing the lactam ring-opened streptothricin derivative and the like.
2. Related Art
Streptothricins (sometimes abbreviated as “ST,” see FIG. 1) are broad-spectrum antibiotics first isolated from Streptomyces lavendulae in 1943 (Waksman, S. A., J. Bacteriol., 46, 299-310 (1943)). All STs include a carbamoylated D-gulosamine to which is attached a β-lysine homopolymer (1 to 7 residues), and the unusual amino acid streptolidine in the amide form (streptolidine lactam). STs are potent inhibitors of protein biosynthesis in prokaryotic cells, and also strongly inhibit the growth of eukaryotic cells such as yeasts, fungi, protozoa, insects and plants. Consequently, they are used as effective selective agents for recombinant DNA work in some of these organisms. However, STs are not used therapeutically due to their nephrotoxicity.
Many ST resistance genes have already been identified in Tn1825, Tn1826 and other transposons isolated from ST-resistant bacteria (Partridge, S. R. & Hall, R. M., J. Clin. Microbiol., 43, 4298-4300 (2005)), and transposons of this kind have also been isolated from clinically problematic pathogens such as Shiga toxin-producing E. coli (Singh, R et al., S., J. Antimicrob. Chemother., 56, 216-219 (2005) and a Shigella strain (Peirano, G et al., J. Antimicrob. Chemother., 55, 301-305 (2005). Bacterial resistance to antibiotics exhibiting protein biosynthesis inhibition activity (such as aminoglycosides) can result from three causes (Vakulenko, S. B. & Mobashery, S. (2003), Clin. Microbiol. Rev. 16, 430-450): (1) decreased antibiotic uptake and accumulation, (2) modification of 16S RNA or ribosomal proteins, and (3) enzymatic modification of the antibiotics. In the case of bacterial resistance to STs, however, so far the only known resistance mechanism is a common resistance mechanism in which the ST molecule is modified by monoacetylation (see FIG. 1) of the β-amino group (position 16) of the β-lysine. In fact, ST-resistance genes encoding N-acetyltransferase (NAT) have also been identified in ST-producers such as Streptomyces lavendulae (Horinouchi, S. et al., J. Bacteriol., 169, 1929-1937 (1987)), Streptomyces rochei (Fernandez-Moreno, M. A., Vallin, C. & Malpartida, F., J. Bacteriol., 179, 6929-6936 (1997)) and Streptomyces noursei (Krugel, H. et al, Gene, 62, 209-217 (1988); Grammel, N., et al., Eur. J. Biochem., 269, 347-357 (2002)), and the roles played by these genes in self-resistance against their own STs are being investigated. On the basis of this resistance mechanism, and the fact that ST-D is more effective than ST-F as an antibiotic against many bacteria, it has been shown that the β-lysine moiety plays a crucial role in antibiotic activity. On the other hand, the groups of Inamori (Inamori, Y. et al., Chem. Pharm. Bull. (Tokyo), 36, 1577-80 (1988)) and Taniyama (Taniyama, H., Sawada, Y. & Kitagawa, T., J. Antibiot. (Tokyo), 24, 662-666 (1971)) have independently reported that ST-F-acid (FIG. 1; racenomycin-A-acid in their work), which was chemically synthesized from ST-F, did not exhibit antibiotic activity against bacteria, fungi and plants. These results confirmed that the streptolidine lactam is also indispensable for antibiotic activity. However, the antibiotic activity of ST-D-acid has not been tested.
Under these circumstances, there is demand for an enzyme capable of reducing the antibiotic activity (that is, toxicity) of streptothricin with respect to eukaryotic cells without sacrificing antibiotic activity with respect to prokaryotic cells, as well as a streptothricin derivative having reduced toxicity with respect to eukaryotic cells while retaining antibiotic activity with respect to prokaryotic cells, and a method for manufacturing this streptothricin derivative.
In an effort to solve the above-described problems, it has been discovered that a protein having the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 has the activity of opening the lactam ring of streptothricin. It has also been discovered that the lactam ring-opened streptothricin D derivative (ST-D-acid) obtained with this enzyme has reduced toxicity with respect to eukaryotic cells while retaining antibiotic activity with respect to prokaryotic cells.